We have recently demonstrated that fibronectin (FN) matrix fibrils are elastic and very extensible. We now propose to determine the mechanism of this elasticity. Our hypothesis is that the elasticity of FN is due to force-induced unraveling of FN-III domains, which has been demonstrated recently for single molecules by atomic force microscopy. We propose to map the elasticity of FN by constructing expression proteins containing small segments or repeated copies of different FN-III domains. We will attempt to make a mutant FN with disulfide bonds in each FN-III domain, greatly reducing its extensibility. If the subsequent matrix fibrils have lost elasticity this will support our hypothesis. This construct will also test an important hypothesis, that stretching is essential for matrix assembly. We propose to investigate quantitatively the FN-integrin bond. We want to extend our recent study, which demonstrated an extensive synergy interface in FN9, by mapping this interface completely. We propose to investigate the binding of mini-integrins, small expression proteins corresponding to the active site of alpha5 and beta1 integrins. We will map their binding sites in FN9-10, and perform quantitative measures of binding affinity. Finally, we will attempt to measure the force that can be supported by a single FN-integrin bond, using a biomembrane force probe. We will compare this force to the 50-200 pN found to unravel FN-III domains in single molecule stretching, to determine how many integrin bonds are needed to stretch FN fibrils. We have recently achieved a conditional FN knockout in mice using the cre/lox approach. We propose to study the role of FN in later embryonic development by knocking the gene out at embryonic day 10 or later (mice die at E8.5 if the knockout is from the beginning). FN is expressed during the development of many organs after E10, so our timed knockout should demonstrate where and how FN is important in organ development.